BMS 100 Lecture 5: Proteins + Enzymes 1 PDF

Lecture 5: Proteins + Enzymes 1 In-class Dr. Rhea Hurnik BMS 100 Outline Proteins Classification Structure: Primary, secondary, tertiary, quaternary Denaturation Enzymes 1 Specificity Mechanisms Acid Base, Covalent catalysis Cofactors and Coenzymes Effect of temperatures and pH Regulation Protein St...

Lecture 5: Proteins + Enzymes 1 In-class Dr. Rhea Hurnik BMS 100 Outline Proteins Classification Structure: Primary, secondary, tertiary, quaternary Denaturation Enzymes 1 Specificity Mechanisms Acid Base, Covalent catalysis Cofactors and Coenzymes Effect of temperatures and pH Regulation Protein Structure During pre-learning we look at the various levels of protein structure § Primary, secondary, tertiary, and quaternary Let’s consider the loss of protein structure – ie disruption in folding/shape § This is called protein denaturation § Occurs when the bonds holding the protein together are disrupted. What are some examples of these bonds? Protein denaturation: General Bonds within proteins can be disrupted and the proteins denatured by using: § Strong acids or bases, or reducing agents: Add or remove hydrogens § Organic solvents, detergents: Disrupt hydrophobic, polar and charged interactions § Salts: Disrupt polar and charged interactions § Heavy metal ions H2 N Salt bridge Protein Denaturation – Heavy Metals Let’s consider how heavy metals will denature proteins § Ex: mercury (Hg+2) and lead (Pb+2) Based on charge, what type of amino acid side chain do you think these heavy metals can bind to? § What type of interaction within a protein would therefore be disrupted? Protein Denaturation – Heavy Metals cont. As well as binding to negatively charged groups, heavy metals can also bind to sulfhydryl (SH) groups § This alters the shape of the protein § This is the basis of “lead poisoning” Pb+2 binds to the SH groups in two enzymes needed for the synthesis of Hb § Lack of Hb synthesis leads to the hallmark anemia associated with lead poisoning In class assignment time Enzymes Enzymes outline: § Specificity § Mechanisms § Role of cofactors and coenzymes § Effect of temperatures or pH § Regulation New topic - Enzymes Enzymes are an example of a type of globular protein. § Most biochemical reactions that occur in our body require enzymes in order to occur at a pace fast enough to support life. We’ve already learned about several enzymes already! § Can you name one from glycolysis? Enzymes introduction Enzymes = protein catalysts that speed up specific reactions and remained unchanged by the reaction § Enzymes speed up a reaction by lowering the activation energy of the reaction without changing: The standard free energy (ΔG) of the reaction The equilibrium of the reaction § They speed up a specific reaction by lowering the activation energy Ea Activation Energy (Ea) The minimal amount of energy needed to make/break the bonds necessary for a reaction to occur § Also known biochemically as the free energy of activation (ΔG‡) § Sometimes defined as the amount of energy needed to reach the transition state (TS) The transition state is the highest energy configuration formed when changing from reactants to products Transient and not isolated Activation Energy (Ea) To note: Free energy (𝚫G‡ ) Progress of reaction § The activation energy is lowered in the catalyzed reaction § No change in overall standard free energy of the equation (ΔG) Enzymes specificity Enzyme molecules contain a special cleft called the active site § The active site forms by precise quaternary structure of the protein § Amino acids in the active site participate in substrate binding and catalysis Enzyme specificity Enzymes are highly specific Only a substrate of the correct size and shape can enter into the active site. § Once inside the site, the substrate binds the enzyme forming an enzyme-substrate (ES) complex § Binding of substrate is thought to induce a conformational change in shape of the enzyme This is called the Induced fit model Enzyme mechanisms Once a substrate binds to the active site, how exactly does it speed up a reaction? The induced-fit between the correct substrate and enzyme allows for: § Electrostatic interactions to form between the two § The correct positioning of catalytic groups in the enzyme Catalytic groups may speed up reactions in two main ways Acid-base catalysis Covalent catalysis Enzyme mechanisms: Acid-base effects Addition or removal of a proton can make a substrate more reactive § The side chains of certain amino acids can add or remove H+’s by acting as general acids or bases Which amino acid side chain do you think can function easily as an acid or a base? Enzyme mechanisms: Acid-base effects Example Enzyme mechanisms: Covalent catalysis A nucleophilic side group in the enzyme active site forms a temporary covalent bond with the substrate § Common nucleophilic side groups include: Asp and Glu (R-COO-) Ser (R-OH) and Cys (R-SH) § Serine and Cystein are only weakly nucleophilic, but their nucleophilicity is enhanced by the presence of other amino acids that can remove the H Enzyme mechanisms: Covalent catalysis Example Cofactors and Coenzymes Enzymes often have help from cofactors and coenzymes § Cofactors are typically metal cations Mg2+, Zn2+ § We have already discussed the role of Mg2+ in several glycolytic enzymes involving ATP. What was the role of magnesium? Cofactors and Coenzymes Enzymes often have help from cofactors and coenzymes § Cofactors are typically metal cations Mg2+, Zn2+ § We have already discussed the role of Mg2+ in several glycolytic enzymes involving ATP. What was the role of magnesium? Magnesium helped to position the ATP in the enzyme active site Helped to stabilize the negative changes on the ATP Cofactors and Coenzymes Enzymes often have help from cofactors and coenzymes § Coenzymes Typically derived from vitamins What are some examples of coenzymes we have learned so far? Cofactors and Coenzymes Enzymes often have help from cofactors and coenzymes § Coenzymes Typically derived from vitamins What are some examples of coenzymes we have learned so far? § B3: NAD+ ßà NADH + H+ Can accept or donate elections in redox reactions Cofactors and Coenzymes In summary, cofactors and coenzymes can help enzymes speed up reactions in three main ways: § Can help position the substrate in the active site of the enzyme § Can stabilize negative charges on the substrate or the TS to make it easier for a nucleophilic attack to occur § Can accept/donate electrons in redox reactions Effect of temperature The optimal temperature for an enzymes is usually the temperature of the organism § Do you think a fevers are good or bad? Effect of pH Changing the pH can change the protonation state of the enzyme and/or the substrate § What types of bonds between E and S would potentially be disrupted by a change in pH? H-bonds – for example: § If an H is removed, no H-bond can be formed § If an H is added, an H-bond might form that is not usually formed. Electrostatic interactions - for example: § Adding an H can turn COO- into COOH § Removing an H can turn NH3+ into NH2 Effect of pH – Thinking question: Consider the lysosome § What is the function of a lysosome? Main site of intracellular enzymatic degradation for a wide range of molecules At what pH do you suppose lysosomal enzymes are active at? (pH of ~7 or pH of ~45?) What would happen to a lysosomal enzyme if it were release into the cytosol Lysosome Enzymes regulation The activity of enzymes can be controlled in four main ways § 1. Genetic § 2. Covalent modification § 3. Allosteric regulation § 4. Compartmentalization Enzymes regulation: Genetic Enzymes transcription can be induced or repressed based on the needs of the cell § In times of needs enzyme transcription and translation will be induced § In times of excess enzyme transcription and translation will be repressed We have already discussed several ways in which this can happen. § What were some examples? Enzymes regulation: Genetic Let’s look at an example: Regular consumption of a meals rich in carbohydrates High insulin Increased transcription of genes for glucokinase, PFK-1, and pyruvate kinase Increased translation Higher amount of glucokinase, PFK-1, and pyruvate kinase in the cytosol More efficient conversion of glucose to pyruvate Enzyme regulation: Covalent modification Involves altering the structure of an enzyme (or “proenzyme”) by making or breaking covalent bonds § There are two types of covalent modification: Reversible Irreversible Enzyme regulation: Covalent modification Reversible covalent modification § Involves addition or removal of a group to the enzyme that causes it to convert to its active or inactive form What is a common group that can do this? § Does addition of this group activate or inactivate the enzyme? Other common groups that can be added to or removed enzymes include methyl and acetyl groups Enzyme regulation: Covalent modification Example – glycogen metabolism § The main regulated enzyme in glycogenesis is deactivated by phosphorylation while the main enzyme in glycogenolysis is activated by phosphorylation. Phosphorylation is catalyzed initially by the same protein, protein kinase A (PKA) This prevents both pathways from running at the same time Enzyme regulation: Covalent modification activated Inhibited Example – glycogen metabolism Protein kinase A ATP Protein kinase A ADP ATP Glycogen phosphorylase kinase Glycogen phosphorylase kinase ATP Glycogen phosphorylase Glycogen synthase P ADP Glycogen synthase ADP Glycogen phosphorylase Glycogenolysis P No glycogenesis P Enzyme regulation: Covalent modification activated Inhibited Example – glycogen metabolism continued Protein kinase A ATP Protein kinase A ADP ATP Glycogen phosphorylase kinase Glycogen phosphorylase kinase ATP Glycogen phosphorylase Glycogen synthase P ADP Glycogen synthase ADP Glycogen phosphorylase Glycogenolysis P No glycogenesis P Enzyme regulation: Covalent modification Irreversible covalent modification § Involves cleavage of peptide bonds in proenzymes or zymogens § Makes sure the enzyme is not used until it is in the correct location and until it is needed Enzyme regulation: Allosteric modification Allosteric modification of allosteric enzymes § Binding to enzyme’s allosteric site changes the conformation and activity of the enzyme Changes the binding affinity of the substrate at the active site § More to come in Enzymes II § Commonly used to control regulatory enzymes Enzyme regulation: Allosteric modification Allosteric enzymes have more than one subunit § Allosteric site is on one subunit, active site on another § The binding of an effector molecule to an allosteric enzyme can either: Increase binding of the substrate to the enzyme § Effector = activator Decrease binding of the substrate to the enzyme § Effector = inhibitor Enzyme regulation: Allosteric modification Consider the glycolytic enzyme phosphofructokinase-1 (PFK-1): What reaction is catalyzed by PFK-1? It is reversible or irreversible? § Phosphofructokinase-1 is inhibited allosterically by high levels of ATP Can you think of why this might be the case? § Phosphofructokinase-1 in activated allosterically by high levels of AMP Can you think of why this might be the case? Enzyme regulation: Compartmentalization Compartmentalization of enzymes via membranebound organelles allows for regulation by: § 1. Separation of enzymes from opposing pathways into different cellular compartments, and selective transportation of substrates Can you think of metabolic pathways that are compartmentalized in different areas of the cell? Enzyme regulation: Compartmentalization Compartmentalization of enzymes via membranebound organelles allows for regulation by: § 2. Creation of unique microenvironments Lysosomal enzymes function at a pH around 4.5-5, while most other cellular enzymes function at a pH around 7.